N-Glycosylation of the alpha subunit does not influence trafficking or functional activity of the human organic solute transporter alpha/beta
© Soroka et al; licensee BioMed Central Ltd. 2008
Received: 07 July 2008
Accepted: 10 October 2008
Published: 10 October 2008
The organic solute transporter (OSTα-OSTβ) is a heteromeric transporter that is expressed on the basolateral membrane of epithelium in intestine, kidney, liver, testis and adrenal gland and facilitates efflux of bile acids and other steroid solutes. Both subunits are required for plasma membrane localization of the functional transporter but it is unclear how and where the subunits interact and whether glycosylation is required for functional activity. We sought to examine these questions for the human OSTα-OSTβ transporter using the human hepatoma cell line, HepG2, and COS7 cells transfected with constructs of human OSTα-FLAG and OSTβ-Myc.
Tunicamycin treatment demonstrated that human OSTα is glycosylated. In COS7 cells Western blotting identified the unglycosylated form (~31 kD), the core precursor form (~35 kD), and the mature, complex glycoprotein (~40 kD). Immunofluorescence of both cells indicated that, in the presence of OSTβ, the alpha subunit could still be expressed on the plasma membrane after tunicamycin treatment. Furthermore, the functional uptake of 3H-estrone sulfate was unchanged in the absence of N-glycosylation. Co-immunoprecipitation indicates that the immature form of OSTα interact with OSTβ. However, immunoprecipitation of OSTβ using an anti-Myc antibody did not co-precipitate the mature, complex glycosylated form of OSTα, suggesting that the primary interaction occurs early in the biosynthetic pathway and may be transient.
In conclusion, human OSTα is a glycoprotein that requires interaction with OSTβ to reach the plasma membrane. However, glycosylation of OSTα is not necessary for interaction with the beta subunit or for membrane localization or function of the heteromeric transporter.
The organic solute transporter (OSTα-OSTβ) is a heteromeric transporter of bile acids and other organic solutes and steroids. In the human, OSTα-OSTβ is found predominantly in epithelial cells of liver, intestine, kidney, adrenal gland and testis. It is expressed on the basolateral membrane of these cells and has been shown to transport estrone 3-sulfate, digoxin, dehydroepiandrosterone 3-sulfate, prostaglandin E2 and a variety of bile acids [1–3]. Regulation of this basolateral transporter is through the action of the bile acid-activated nuclear receptor, the farnesoid × receptor (FXR) . Thus, conditions of cholestasis have been shown to result in up-regulation of OSTα-OSTβ at both the mRNA and protein levels . Recently the importance of this transporter in intestinal bile acid transport and in the enterohepatic circulation has been confirmed in Ostα-/- mice . Data from studies of these mice highlight the role of Ostα-OSTβ and FGF15 in regulating hepatic bile acid synthesis.
It was noted early on that transport activity required the coexpression of two distinct gene products. The first, Ostα, is a predicted 340-amino acid protein with seven membrane spanning domains and the second, Ostβ, is a 128-amino acid, single membrane spanning protein . Transport is bidirectional across the plasma membrane, and most likely occurs by facilitated diffusion of substrates down their electrochemical gradients . Plasma membrane localization and functional activity requires the expression of both subunits [3, 6–8]. Several groups have shown that the functional requirement for co-expression of both subunits is associated with the physical association of the two proteins [7, 8]. Dawson and colleagues demonstrated that mouse Ostβ was necessary for mouse Ostα to acquire N-glycosylation in transfected HEK293 cells, thus suggesting that the beta subunit is acting as a chaperone to allow the alpha subunit to exit the ER . Quality control at the level of the ER can involve many different mechanisms. Newly synthesized proteins must be folded correctly and, in some cases, must be assembled into multimeric protein complexes in order to be trafficked to the Golgi and plasma membrane. If this does not occur the protein may be ubiquitinated and designated for degradation. Thus, the chaperone activity of OSTβ may require a properly folded alpha subunit or may aid in the folding of the peptide. Alternatively, the protein-protein interaction may mask a retention/retrieval motif or reveal a forward trafficking motif in the alpha subunit. Recent work shows that both subunits must be expressed in order to prevent degradation of the other subunit, suggesting a specific interaction between the two proteins [5, 7]. Sun and colleagues have suggested that OSTβ is interacting with the N-terminus, and not the C-terminus, of OSTα . This raises the question of whether the glycosylation of the alpha subunit could influence the interaction with the beta subunit and, thus, affect membrane localization and function of the intact transporter. Therefore, in this study we have sought to examine more fully the interaction of OSTα and OSTβ in two mammalian expression systems where we can look at both the endogenous and exogenous, transfected expression of the human transporter.
The human hepatocellular carcinoma cell line, HepG2, and the monkey kidney cell line, COS7, were acquired from ATCC (Manassas, VA). HepG2 cells were cultured in MEM with non-essential amino acids (ATCC) containing 10% FBS and 1% penicillin-streptomycin, at 37°C with 5% CO2. COS7 cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin, at 37°C with 5% CO2.
After HepG2 cells reached ~70% confluence, they were washed and cultured in fresh medium containing 10% charcoal-stripped serum in the presence or absence of 50 μM chenodeoxycholate (CDCA) (Sigma, St Louis, MO), or 2 μM 6-ethyl CDCA (Dr. Roberto Pellicciari, Universita Di Perugia, Italy). Twenty four to forty-eight hours after addition of CDCA, RNA and protein were isolated or cells were fixed for immunofluorescence as described below. To inhibit glycosylation, tunicamycin (Sigma) was added at concentrations indicated in the figure legends 6 hrs after the addition of CDCA treatment in HepG2 cells or 4 hrs after the initiation of transfection in COS7 cells.
Lysates from COS7 cells transfected for 48 hrs with OSTα-FLAG and OSTβ-MYC were digested with peptide:N-glycosidase F (PNGase F) and endoglycosidase H (EndoH) according to the manufacturer's instructions (New England Biolabs) and subjected to SDS-PAGE as described below.
Cloning human OST alpha, beta and vector constructs
HepG2 cell cDNA was used as a template. We generated a 1.03 kb cDNA fragment encoding the full-length of human OSTα and a 0.4 kb cDNA fragment encoding the full-length of human OSTβ by PCR. The primers for amplification of human OSTα and OSTβ were based on the published human sequences (GenBank accession number AK172837 and AY194242). The forward primer OSTα 5'-GCTTGGTACCATGGAGCCGGGCAGGACCCAGATAA-3' and the reverse primer OSTα 5'-CCGCTCGAGTTACTTGTCATCGTCGTCCTTGTAATCCCCGGCTTTGAGGTTCAAGTCCAGGTC-3' were used. The forward primer OSTβ 5'-GCTGGATCCACCATGGAGCACAGTGAGGGGGCTCC-3' and the reverse primer OSTβ 5'-GCACTCGAGGCTCTC AGTTTCTGGTACATCCGG-3' were used. The amplified cDNA fragment encoding the full-length of OSTα was then subcloned into the Kpn I and Xho I sites of the mammalian expression vector pcDNA3.1 (Invitrogen) and the cDNA fragment encoding the full-length of OSTβ was subcloned into the BamH I and Xho I sites of pcDNA3.1 Myc/His vector (Invitrogen). PcDNA3.1-OSTα-FLAG and pcDNA3.1-OSTβ-Myc/His were sequenced using Yale Keck DNA sequencing facility. The coding sequences were identical to the published sequences with the GenBank accession numbers for OSTα [AY194243] and for OSTβ [BC103842].
COS7 cells were transfected with FuGene 6 (Roche) using 1 μg OSTα-FLAG or OSTβ-Myc DNA/9 cm2 surface area, according to manufacturer's instructions. pcDNA vector control (1 μg DNA) was used when only one subunit was transfected. Cells were harvested 24-48 hr after transfection, as described for Western blotting or immunofluorescence.
Cells were extracted with Trizol (Invitrogen, Carlsbad, CA) and RNA was isolated according to manufacturer's instructions. Quantitative RT-PCR was carried out as described previously  using Applied Biosystems 7500 DNA sequence detector system with TaqMan universal master mix (Applied Biosystems, Foster City, CA). Specific primer pairs for hOSTα and hOSTβ were the same as previously described .
Cells were washed with PBS and then extracted directly in RIPA buffer (25 mM Tris, pH 7.2, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS) or in 1% Triton X-100, 50 mM Tris HCl, pH 7.4,150 mM NaCl, 1 mM EDTA for immunoprecipitation. Lysates were centrifuged at 10,000 × g for 20 min and the supernatant was collected for analysis using SDS-PAGE. Immunoprecipatation was performed using anti-FLAG affinity gel (M2, Sigma, St Louis, MO) or anti-Myc polyclonal antibody (abcam, Cambridge, MA) and Protein A/G beads (Santa Cruz). Lysates were precleared and negative controls were performed with non-specific anti-mouse IgG. In the case of immunoprecipitation of endogenous protein from HepG2 cells, a Native IgG kit from Pierce was used with polyclonal antibodies raised against OSTα (hOSTα-327) and OSTβ (hOSTβ-1) provided by Ned Ballatori (Rochester, NY).
Pulse-chase experiments were carried out in COS7 cells 24 hr after transfection. After incubation for 15 min with Cys/Met minus media, cells were pulsed for 15 min with media containing 135 μCi 35S Trans-label (MP Biomedicals, Solon, OH). Cells were either extracted immediately in 1% Triton X-100, 50 mM Tris Hcl, pH 7.4,150 mM NaCl, 1 mM EDTA or chased for 2 hr in complete media. Immunoprecipitation was carried out as described above.
Cells were fixed with cold methanol for 10 min or with 4% paraformaldehyde for 15 min. Quenching of non-specific fluorescence in formaldehyde fixed cells was done with 50 mM NH4Cl for 20 min prior to blocking 20 minutes in blocking buffer (PBS, 1% BSA, 0.05% Triton X-100). In the case of OSTβ, non-permeablized conditions using no detergent was found to give better surface labeling. Primary antibody was diluted in blocking buffer and incubated on the cells for 2 hours at room temperature. After washing in PBS, secondary antibody (Alexa 594 or 488 anti-IgG (Invitrogen)) was incubated for 1 hour at room temperature. Fluorescence was visualized with a Zeiss LSM510 (Carl Zeiss Inc, Thornwood, NY) confocal microscope and images processed with Photoshop (Adobe, Mountainview, CA).
HepG2 cells were cultured in 35 mm dishes as described above. At ~70% confluency 50 μM CDCA or vehicle was added and the culture continued for 48 hrs. 3H-Taurocholate (1 μM) or 3H-estrone 3-sulfate (15 nM) were made up in transport buffer (116 mM NaCl, 5.3 mM KCl, 1.1 mM KH2PO4, 0.8 MgSO4.7H2O, 1.8 mM CaCl, 11 mM glucose, 10 mM HEPES) and warmed to 37°C. For each time point, triplicate dishes were washed 3 times with warm transport buffer alone and then incubated for the given time with 1 ml transport buffer containing 3H-substrate. Uptake of substrates was stopped by rapid addition and aspiration of 1 ml of cold transport buffer three times. Cells were lysed with 1 ml 0.5% Triton X100. Cell lysates (600 μl) were combined with OptiFluor scintillation fluid (5 ml) and counted in a PerkinElmer WinSpectral LSC (PerkinElmer, Waltham, MA). Protein content of the lysates was determined with the BCA reagent (Pierce Biotechnology, Rockford, IL) and used to normalize the counts.
N-glycosylation of OSTα is not required for plasma membrane expression in HepG2 cells
Plasma membrane expression of OSTα requires OSTβ, but not N-glycosylation
The glycosylation status of OSTα was further clarified by treatment of cell lysates with the glycosidases, Endo H and PNGase F. These two enzymes can distinguish between N-glycans that only contain the core oligosaccharide that has been added in the ER (Endo H sensitive) and those that have trafficked through the Golgi and have had their carbohydrate chains modified (PNGase F sensitive, Endo H resistant) . Figure 4B shows that the 40 kD band was sensitive to PNGase F, but not Endo H, treatment, indicating that the mature alpha subunit has exited the Golgi. The 35 kD band was sensitive to both Endo H and PNGase F and, thus, represents a glycoprotein that has not trafficked through the Golgi. The 31 kD band remains after both glycosidase treatments, confirming that it represents the non-glycosylated OSTα subunit. These data demonstrate that human OSTβ is required for human OSTα to be processed from the high mannose type N-linked glycan in the ER to complex oligosaccharides in the cis/medial Golgi region. Immunofluorescence of tunicamycin treated COS7 cells transfected with OSTαFLAG and OSTβ-Myc showed that the lack of glycosylation did not prevent OSTα trafficking to the plasma membrane, confirming data seen in HepG2 cells (Figure 4C). Thus, the interaction of the beta subunit with the alpha subunit in the ER and the subsequent trafficking through the Golgi does not require that OSTα be glycosylated.
Immunoprecipitation demonstrates that immature forms of OSTα and OSTβ associate
The importance of the novel heteromeric, basolateral transporter, Ostα-Ostβ, in enterohepatic circulation of bile acids and the homeostasis of bile acid synthesis has recently been confirmed . Although it is clear that function of this facilitated transporter requires expression of both subunits, it is not known whether functional activity depends upon (1) the acquisition of N-glycosylation of the alpha subunit, (2) the beta subunit for its ability to release the alpha subunit from an ER retention signal, or (3) the physical interaction of the two proteins at the plasma membrane. The data provided here indicate that glycosylation of OSTα is not necessary for transporter localization or function. Furthermore, it shows that the physical interaction of the two subunits may be transient, suggesting that association at the plasma membrane may not be necessary for transporter function.
Glycosylation of a protein is one of the major biosynthetic functions of the ER and is a common post-translational modification of membrane proteins. Although the addition of the "core" oligosaccharide occurs in the ER, further extensive processing or trimming occurs in the Golgi and results in what is commonly referred to as the complex or mature glycoprotein . N-glycosylation is found usually in the sequences Asn-X-Ser or Asn-X-Thr, where X is any amino acid [13, 14]. Although this consensus motif is found in the N-terminus of the alpha subunit in the mouse, rat and skate, it is not present in the human OSTα . Instead, the sole asparagine residue in an extracellular site is in the sequence Asn25-X-Gly in the N-terminus. We have shown in this study that, despite the lack of traditional consensus sequence, human OSTα is expressed on the cell surface as a glycoprotein. Similar to previous reports [3, 4, 6, 7, 15] our data indicate that endogenous alpha subunit migrates in SDS-PAGE as a single band and precursor forms are not detected. This suggests that in the presence of the beta subunit the glycoprotein is efficiently trafficked through the Golgi. It is only in the over-expressing transfected cells that the multiple forms of the alpha subunit are seen (Figure 3, 4 and 5 this manuscript; [3, 6].
The necessity for glycosylation of proteins has been studied for many years and is largely believed to be important in proper folding and stabilization of newly synthesized proteins and in affecting the charge and solubility of the protein [16, 17]. The critical nature of this folding is highlighted by the finding that detection of misfolded glycoproteins in the ER can result in ER-associated degradation (ERAD) [18, 19]. Our data indicate that the lack of oligosaccharide chain on the alpha subunit does not designate the polypeptide for ERAD. Instead, after tunicamycin inhibition of glycosylation, the transporter was still trafficked properly to the plasma membrane where it was fully functional, indicating that interaction between the alpha and beta subunits is not compromised by the lack of oligosaccharide. Perhaps because the alpha subunit of the organic solute transporter has only one asparagine residue in an extracellular domain, the affect of the absence of the carbohydrate on folding is not critical. Tunicamycin treatment has been used to study glycosylation of other hepatocyte proteins. The absence of oligosaccharide did not affect the secretion of transferrin or very low density lipoprotein, but did interfere with the ability of the apical membrane protein, Mrp2, to be trafficked to the plasma membrane in rat hepatocytes . And recently the N-linked carbohydrates have been described for the hepatocyte basolateral membrane protein oatp1a1 and found to be important in the protein's localization and function . In HepG2 cells it has been reported that five of eight glycoproteins studied did not require glycosylation for their trafficking . Mochizuki et al have shown that rat Bsep requires at least two of its four N-linked glycans for proper protein stability, intracellular trafficking and functional activity .
Interestingly, we (Figure 5B) and others [5, 7] have shown that the absence of one of the subunits leads to degradation of the other subunit. Thus, it is the presence and interaction of the two subunits that are critical to the stability of the heteromeric, intact transporter, and not the glycosylation of the alpha subunit. Protein-protein interactions in the ER are known to be critical for many different processes, including trafficking and function of multimeric membrane proteins. The presence of fully functional oligomeric complexes at the plasma membrane can involve specific ER retention/retrieval motifs[24, 25], anterograde ER export signals [26, 27], interaction with scaffold protein [28–31], and phosphoylation [29, 32]. The necessity for interaction between OSTα and OSTβ subunits in the ER suggests that physical association of the two proteins may mask a retention/retrieval motif or, alternatively, may reveal a forward trafficking motif. The RXR motif is one such retention/retrieval sequence and it is interesting that both the alpha and beta subunits contain an RXR-like motif in their C-terminal sequence. It remains to be determined whether this sequence is important in the localization of the organic solute transporter.
Our immunoprecipitation data confirm that the OSTα and OSTβ interaction is essential early in the biosynthetic process, but suggest that it may not be necessary later once the major protein gets to the plasma membrane. Because the only way to get OSTα to the plasma membrane is to co-express the beta subunit, it is impossible to determine if the alpha subunit actually requires the beta subunit for its functional activity. However, the lack of co-precipitation between the mature form of OSTα and the OSTβ subunit suggests that this may not be the case. When Li and colleagues  performed similar immunoprecipitations in HEK293 cells transfected with mouse Ostα and Ostβ constructs, they also saw only a single band after precipitation with anti-Myc. However, they indicate that it is the mature form of the protein. Given that all data point to the interaction of the subunits in the ER, one would also expect to see the immature form precipitated. Similarly, in mouse ileum Li et al show only one band for Ostα on Western blots and this protein is co-precipitated by an antibody to Ostβ . Although the explanation for these differences in immunoprecipitation is still unclear, we cannot discount that it is due to species variability or species-specific antibody affinity.
The possible transient nature of the subunit interaction also appears to be in conflict with immunofluorescent studies which suggest co-localization of the subunits at the plasma membrane in transfected cells (Figure 2 and . However, the finding of a yellow color indicating co-localization may be due to the close proximity of the two subunits, not the actual association. Optical microscopes are unable to resolve two items that are closer together than 200 nm. Also, we cannot discount the possibility that, similar to tunicamycin treated cells, some "immature" protein might be expressed on the plasma membrane, and, thus, be detected by the primary antibodies. Bimolecular fluorescence complementation has also been used to study the interaction of the two subunits in HEK293 cells transfected with mouse Ostα and Ostβ . These studies clearly show that complementation occurs between Ostα and Ostβ and results in plasma membrane localization. However, the possibility that the interaction might be transient cannot be assessed because, once the complementation reaction occurs, it is irreversible.
In conclusion, this study demonstrates that, although human OSTα is a glycoprotein, the carbohydrate chains are not necessary for interaction with OSTβ or subsequent exit from the ER. Furthermore, plasma membrane localization and functional activity of the organic solute transporter does not depend upon N-glycosylation. Interaction between the two subunits occurs early in the biosynthetic pathway, but may not be necessary at the plasma membrane.
- Endo H:
farnesoid × receptor
organic solute transporter
- PNGase F:
We would like to thank Ned Ballatori for antibodies to OSTα and OSTβ and for helpful discussions. This work was supported by National Institutes of Health grants DK 25636 to JLB and the Yale Liver Center DK P30 34989.
- Seward DJ, Koh AS, Boyer JL, Ballatori N: Functional Complementation between a Novel Mammalian Polygenic Transport Complex and an Evolutionarily Ancient Organic Solute Transporter, OSTα-OSTβ. J Biolo Chem. 2003, 278 (30): 27473-27482. 10.1074/jbc.M301106200.View ArticleGoogle Scholar
- Wang W, Seward DJ, Li L, Boyer JL, Ballatori N: Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate. PNAS. 2001, 98: 9431-9436. 10.1073/pnas.161099898.PubMed CentralView ArticlePubMedGoogle Scholar
- Ballatori N, Christian WV, Lee JY, Dawson PA, Soroka CJ, Boyer JL, Madejczyl MS, Li N: OSTa-OSTb: A Major Basolateral Bile Acid and Steroid Transporter in Human Intestinal, Renal, and Biliary Epithelia. Hepatology. 2005, 42: 1270-1279. 10.1002/hep.20961.View ArticlePubMedGoogle Scholar
- Boyer JL, Trauner M, Mennone A, Soroka CJ, Cai S-Y, Mounstafa T, Zollner G, Lee JY, Ballatori N: Upregulation of a Basolateral RXR-dependent bile acid efflux transporter OSTα-OSTβ in Cholestasis in Humans and Rodents. Am J Physiol Gastrointest Liver Physiol. 2006, 290: G1124-G1130. 10.1152/ajpgi.00539.2005.View ArticlePubMedGoogle Scholar
- Rao A, Haywood J, Craddock AL, Belinsky MG, Kruh GD, Dawson PA: The organic solute transporter α-β, Ostα-Ostβ, is essential for intestinal bile acid transport and homeostasis. Proc Natl Acad Sci U S A. 2008, 105 (10): 3891-3896. 10.1073/pnas.0712328105.PubMed CentralView ArticlePubMedGoogle Scholar
- Dawson PA, Hubbert HJ, Craddock AL, Zeranque N, Christian WV, Ballatori N: The heteromeric organic solute transporter alpha-beta, Ostα-Ostβ, is an ileal basolateral bile acid transporter. J Biol Chem. 2005, 280: 6960-6968. 10.1074/jbc.M412752200.PubMed CentralView ArticlePubMedGoogle Scholar
- Li N, Cui Z, Fang F, Lee JY, Ballatori N: Heterodimerization, trafficking and membrane topology of the two proteins, Ostα and Ostβ, that constiture the organic solute and steroid transporter. Biochem J. 2007, 407: 363-372. 10.1042/BJ20070716.PubMed CentralView ArticlePubMedGoogle Scholar
- Sun A-Q, Balasubramaniyan N, Xu K, Liu CJ, Ponamgi VM, Liu H, Suchy FJ: Protein = protein interactions and membrane localization of the human organic solute transporter. Am J Physiol Gastrointest Liver Physiol. 2007, 292: G1586-1593. 10.1152/ajpgi.00457.2006.View ArticlePubMedGoogle Scholar
- Jung D, Podvinec M, Meyer UA, Mangelsdorf DJ, Fried M, Meier PJ, Kullak-Ublick GA: Human Organic Anion Transporting Polypeptide 8 Promoter is Transactivated by the Farnesoid × Receptor/Bile Acid Receptor. Gastroenterology. 2002, 122: 1954-1966. 10.1053/gast.2002.33583.View ArticlePubMedGoogle Scholar
- Elbein AD: The tunicamycins-useful tools for studies on glycoproteins. Trends in Biochem Sci. 1981, 6: 219-221. 10.1016/0968-0004(81)90080-3.View ArticleGoogle Scholar
- Lee TK, Koh AS, Cui Z, Peierce RH, Ballatori N: N-glycosylation controls functional activity of Oatp1, an organic anion transporter. Am J Physiol Gastrointest Liver Physiol. 2003, 285: G371-G381.View ArticlePubMedGoogle Scholar
- Wang P, Hata S, Xiao Y, Murray JW, Wolkoff AW: Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains. Am J Physiol Gastrointest Liver Physiol. 2008, 294: G1052-G1059. 10.1152/ajpgi.00584.2007.View ArticlePubMedGoogle Scholar
- Helenius A, Aebi M: Intracellular functions of N-linked glycans. Science. 2001, 291: 2364-2369. 10.1126/science.291.5512.2364.View ArticlePubMedGoogle Scholar
- Bause E: Structural requirements of N-glycosylation of proteins. Biochem J. 1983, 209: 331-336.PubMed CentralView ArticlePubMedGoogle Scholar
- Frankenberg T, Rao A, Chen F, Haywood J, Shneider BL, Dawson PA: Regulation of the Mouse Organic Solute Transporter α-β, Ostα-Ostβ, by Bile Acids. Am J Physiol Gastrointest Liver Physiol. 2005, 290: G912-G922. 10.1152/ajpgi.00479.2005.View ArticlePubMedGoogle Scholar
- Paulson JC: Glycoproteins: what are the sugar chains for?. Trends in Biochem Sci. 1989, 14: 272-276. 10.1016/0968-0004(89)90062-5.View ArticleGoogle Scholar
- Varki A: Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993, 3: 97-130. 10.1093/glycob/3.2.97.View ArticlePubMedGoogle Scholar
- Klausner RD, Sitia R: Protein degradation in the endoplasmic reticulum. Cell. 1990, 62: 611-614. 10.1016/0092-8674(90)90104-M.View ArticlePubMedGoogle Scholar
- Plemper RK, Wolf DH: Retrograde protein translocation: ERADication of secretory proteins in health and disease. Trends in Biochem Sci. 1999, 24: 266-270. 10.1016/S0968-0004(99)01420-6.View ArticleGoogle Scholar
- Struck DK, Siuta PB, Lane MD, Lennarz WJ: Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. J Biol Chem. 1978, 253: 5332-5337.PubMedGoogle Scholar
- Zhang P, Tian X, Chandra P, Brouwer KLR: Role of glycosylation in trafficking of Mrp2 in sandwich-cultured rat hepatocytes. Mol Pharmacology. 2005, 67 (4): 1334-1341. 10.1124/mol.104.004481.View ArticleGoogle Scholar
- Newton SA, Yeo K-T, Yeo T-K, Parent JB, Olden K: Vertebrate Lectins. Edited by: Olden, Parent JB. 1987, New York: Van Nostrand Reinhold, 211-226.Google Scholar
- Mochizuki K, Kagawa T, Numari A, Harris MJ, Itoh J, Watanabe N, Mine T, Arias IM: Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) inMCDK II cells. Am J Physiol Gastrointest Liver Physiol. 2007, 292: G818-G828. 10.1152/ajpgi.00415.2006.View ArticlePubMedGoogle Scholar
- Margeta-Mitrovic M, Jan Y-N, Jan LY: A trafficking checkpoint controls GABAB receptor heterodimerization. Neuron. 2000, 27: 97-106. 10.1016/S0896-6273(00)00012-X.View ArticlePubMedGoogle Scholar
- Zeranque N, Schwappach B, Jan LY: A new ER trafficking signal regulated the subunit stoichiometry of plasma membrane KATP channels. Neuron. 1999, 27: 537-548. 10.1016/S0896-6273(00)80708-4.View ArticleGoogle Scholar
- Ma D, Zeranque N, Lin Y-F, Collins A, Yu M, Jan YN, Jan LY: Role of ER export signals in controlling surface potassium channel numbers. Science. 2001, 291: 316-319. 10.1126/science.291.5502.316.View ArticlePubMedGoogle Scholar
- Stockklausner C, Ludwig J, Ruppersburg J, Klocker N: A sequence motif responsible for ER export and surface expression of Kir2.0 inward rectifier K+ channels. FEBS Lett. 2001, 493: 129-133. 10.1016/S0014-5793(01)02286-4.View ArticlePubMedGoogle Scholar
- Standley S, Roche KW, McCallum J, Sans N, Wenthold RJ: PDZ domain suppression of an ER retention signal in NMDA receptor NR1 splice variants. Neuron. 2000, 28: 887-898. 10.1016/S0896-6273(00)00161-6.View ArticlePubMedGoogle Scholar
- Xia H, Homby Z, Malenka R: An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits. Neuropharm. 2001, 41: 714-723. 10.1016/S0028-3908(01)00103-4.View ArticleGoogle Scholar
- Tiffany AM, Manganas LN, Kim E, Hsueh Y-P, Sheng M, Trimmer JS: PSD-95 and SAP97 exhibit distinct mechanisms for regulating K+ channel surface expression and clustering. J Cell Biol. 2000, 148: 147-157. 10.1083/jcb.148.1.147.PubMed CentralView ArticlePubMedGoogle Scholar
- Fernandez-Larrea J, Merlos-Suarez A, Urena JM, Baselga J, Arribas J: A role for a PDZ protein in the early secretory pathway for the targeting of pro-TGFa to the cell surface. Mol Cell. 1999, 3: 423-433. 10.1016/S1097-2765(00)80470-0.View ArticlePubMedGoogle Scholar
- Scott DB, Blanpied TA, Swanson GT, Zhang C, Ehlers MD: An NMDA receptor ER retention signal regulated by phosphorylation and alternative splicing. J Neurosci. 2001, 21 (9): 3063-3072.PubMedGoogle Scholar
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